1. Field of the Invention
The present invention relates to a method for manufacturing polyhydroxyalkanoate-containing structure comprising the steps of immobilizing on a base material polyhydroxyalkanoate synthase involved in polyhydroxyalkanoate biosynthesis reaction and coating at least part of the base material with polyhydroxyalkanoate by polymerizing a 3-hydroxyacyl coenzyme A by use of the enzyme to synthesize the polyhydroxyalkanoate. More particularly, the invention relates to a method for manufacturing polyhydroxyalkanoate-containing structure by immobilizing to a base material polyhydroxyalkanoate synthase containing an amino acid sequence capable of binding to the base material.
The present invention relates to a structure that has polyhydroxyalkanoate, base material and polyhydroxyalkanoate synthase immobilized on the base material, the polyhydroxyalkanoate coating at least part of the base material. The structure of the present invention encompasses a granular structure (hereinafter called a “capsular structure”) in which polyhydroxyalkanoate is coated on a granular base material, and a plate- or film-like structure (hereinafter called a “laminated structure”) in which at least part of a plate- or film-like base material is coated with polyhydroxyalkanoate.
The structure of the present invention can find a wide range of applications as a functional structure. For example, the capsular structure can have a large number of applications as a variety of functional structures such as a pigment dispersant of excellent dispersion stability and a toner for electrophotography of excellent electrostatic property, and the laminated structure as various functional structures including an OHP film and an electronic device.
2. Related Background Art
Polymeric materials are essential to modern industries and our lives. The materials, which are inexpensive and lightweight and have good moldability, are widely utilized as packaging material and cushioning material, or fiber material, as well as boxes for household electrical appliances. On the other hand, diverse functional materials such as a liquid crystal material and a coat agent are also obtained by utilizing stable properties of these polymeric materials to thereby place substituents of exhibiting various functions on molecular chains of the polymers. These functional materials are higher in added values than polymers for structural materials and thus can be expected to have large market needs even in a small amount. These functional polymeric materials have been produced so far by organic, synthetic chemical methods in synthetic processes of polymers or by modifying synthesized polymers with substituents. Polymers of basic frameworks for functional polymeric materials have been obtained from petroleum based raw material by organic, synthetic chemical methods in most cases. Typical examples of these polymers include polyethylene, poly(ethylene terephthalate), polyesters, polystyrene, poly(vinyl chloride) and polyacrylamides.
Incidentally, the present inventors have focused on a multilayered structure, the base material of the structures being coated with a polymeric compounds as a basic element that imparts large added values to the polymeric compound. A composite structure of extremely useful functionality can be obtained by coating a specific base material with a polymeric compound.
While polymeric compounds used for coating base materials are conventionally synthesized and made to be structures by organic synthetic processes and then various functions are added to them, recently, the production of polymeric compounds by bioengineering approaches has been actively studied and part of it is operational. Known examples include as polymeric compounds derived from microbes polyhydroxyalkanoates (hereinafter sometimes abbreviated as PHAs) such as poly-3-hydroxy-n-butyric acid (hereinafter sometimes abbreviated as PHB), and a copolymer of 3-hydroxy-n-butyric acid and 3-hydroxy-n-valeric acid (hereinafter sometimes abbreviated as PHB/V), polysaccharides such as bacteria cellulose and pullulan, and polyamino acids such as poly-γ-glutamic acid and polylysine. In particular, PHAs can be utilized for various products by melt processing, or the like, like conventional plastics and also exhibit excellent biocompatibility, thus being expected to find applications including flexible materials for medical treatment.
Recently, an attempt has been started to synthesize PHAs in vitro by taking the aforementioned PHB synthase or PHA synthase out of the microbe.
For example, a PHB composed of a 3-hydroxy-n-butyric acid unit has been successfully synthesized by causing the action of 3-hydroxybutylyl CoA on a PHB synthase derived from Alcaligenes eutrophus (Proc. Natl. Acad. Sci. USA, 92, 6279-6283, 1995). In addition, PHBs composed of a 3-hydroxy-n-butyric acid unit or a 3-hydroxy-n-valeric acid unit has been successfully synthesized by causing the action of 3-hydroxybutyryl CoA or 3-hydroxyvaleryl CoA on a PHB synthase derived from Alcaligenes eutrophus (Int. J. Biol. Macromol., 25, 55-60, 1999). Furthermore, in this study, a PHB composed only of the R form of a 3-hydroxy-n-butyric acid unit was synthesized, due to stereo-selectivity of an enzyme, by the action of a racemic modification of 3-hydroxybutyryl CoA. A PHB has been synthesized in vitro using a PHB synthase derived from Alcaligenes eutrophus as well (Macromol. Rapid Commun., 21, 77-84, 2000).
In addition, a PHB composed of a 3-hydroxy-n-butyric acid unit has been successfully synthesized by causing the action of 3-hydroxybutyryl CoA on a PHB synthase derived from Chromatium vinosum (FEMS Microbiol. Lett., 168, 319-324, 1998).
A PHA composed of 3-hydroxydecoic acid unit has been synthesized by causing the action of 3-hydroxydecanoil CoA on Pseudomonas aeruginosa of PHA synthases (Appl. Microbiol. Biotechnol., 54, 37-43, 2000).
As discussed above, application of bioengineering approaches to polymeric compounds will be able to synthesize new polymeric compounds that are difficult to synthesize by conventional organic synthetic methods and provide new functions and structures. In addition, although conventional, organic, synthetic chemical methods requires a manufacturing step of many stages, the bioengineering method needs only a one-stage step in many cases and therefore is expected to simplify the manufacturing step, save costs and shorten the turnaround time. Further, the method makes it possible to decrease the use of organic solvents, acids and alkalis, surfactants, etc., set mild reaction conditions and synthesize a target material from nonpetroeum-based raw material and low purity raw material, thereby being able to realize a synthetic process of a lower environmental load and a resource recycling type. Additionally, for more detailed description of the synthesis of the low purity raw material, the bioengineering synthetic process generally has a high substrate specificity of an enzyme, or a catalyst, which permits a target reaction to selectively proceed even though a material of a low purity is used, thus enabling the use of waste and recycling raw material.
On the other hand, as described previously, the present inventors have focused attention on a structure made by coating a base material with a polymeric compound as an element for imparting a large added value to the polymeric compound. Coating a specific base material with a polymeric compound like this can provide a composite structure having extremely useful functionality. In particular, if this type of structure can be produced by a bioengineering approach as previously mentioned, utilization of a novel polymeric compound that is difficult to produce by a conventional organic synthetic method or new additions of functions and structures will be made possible and thereby a manufacturing process of a lower environmental load and resource recycling type will be realized at a low cost. For example, use of extremely precise molecule recognition ability and stereo selectivity that are specific in catalytic action of living organisms can produce by a simple and easy process of a lower environmental load a novel polymeric compound of functionality that is difficult to produce by a conventional organic synthetic chemical method, or a capsular structure or laminated structure that is coated with an extremely high chirality polymeric compound.
Therefore, it is an object of the present invention to provide a polymeric compound structure of high functionality by means of a bioengineering approach and a manufacturing method thereof and more specifically to provide more effective utilization of an enzyme when a structure, the base material of which is coated with a PHA, is to be produced by taking a PHB synthase or PHA synthase out of the microbe to synthesize a PHA in vitro. In addition, it is another object of the present invention to provide a structure, the base material of which is coated with a polymeric compound, that can be widely utilized as a composite structure of functionality, and an effective manufacturing method thereof.